Five new sesquiterpenoids from agarwood of Aquilaria sinensis

Five new eudesmane-type sesquiterpenoids (aquisinenoids F–J (1–5)) and five known compounds (6–10) were isolated from the agarwood of Aquilaria sinensis. Their structures, including absolute configurations, were identified by comprehensive spectroscopic analyses and computational methods. Inspired by our previous study on the same kinds of skeletons, we speculated that the new compounds have anticancer and anti-inflammatory activities. The results did not show any activity, but they revealed the structure–activity relationships (SAR).


Introduction
Agarwood is the resinous wood of the Aquilaria species of the Thymelaeaceae family [1]. It is a precious traditional Chinese medicinal material and a kind of natural fragrance that is widely distributed in China, India, the Middle East, and Southeast Asia [2]. Agarwood has been considered to play an important role in both traditional Chinese medicine and Ayurvedic medicine in the treatment of stomach disorders, coughs, asthma, sedation, analgesia, and antiemetic [3,4]. Previous studies have shown that 2-(2-phenylethyl)chromones and sesquiterpenes are the characteristic and main bioactive components of agarwood [5,6]. Various bioactivities, including neuroactive [4], gastrointestinal modulation [7], cytotoxicity [8], antibacterial [9], anti-fungal, acetylcholinesterase inhibition [8], anti-inflammatory [10], antiasthmatic [11], antidiabetic [12], and antioxidant [13] activities, have been reported for agarwood extracts [14,15]. Our group recently reported five structurally intriguing and biologically active sesquiterpene dimers [16], which attracted our interest to gain deep insight into novel molecules with effective bioactivities from agarwood. Therefore, the continued study of Aquilaria sinensis has led to the isolation of ten sesquiterpenoids, including five new eudesmane-type sesquiterpenoids ( Figure 1). Herein, we describe the isolation, structural elucidation, and bioactivity evaluation of the new compounds.
Compound 1 was obtained as pale yellow gum, and its molecular formula was inferred from the positive HRESIMS at m/z 273.1464 [M + Na] (Table 1) spectra indicate 15 carbons, including one methyl, eight methylenes (one sp 2 ), one methine, and four nonprotonated carbons (including three sp 2 and one sp 3 ). The planar structure of 1 was mainly constructed by 2D NMR analysis. First, the 1 H, 1 H-COSY spectrum ( Figure 2) displays the correlations of H-1/H-2/H-3 and H-6/H-7/H-8/H-9, suggesting the existence of two spin systems. The HMBC correlations ( Figure 2) of H-1/C-5 (δ C 148.3), H-3/C-4 (δ C 126.8), C-5 and H 3 -15/C-1 (δ C 40.6), C-5, and C-10 (δ C 36.2) indicate the presence of a six-membered ring. Additional HMBC correlations of H-6/C-4, C-5, H-7/C-5, and H 3 -15/C-9 (δ C 43.0) allowed us to assign another six-membered ring, as shown in Figure 2. Furthermore, the positions of a methyl group at C-10 were clarified by the HMBC correlations of H 3 -15/C-1, C-5, C-9, and C-10, respectively. In addition, the HMBC correlations of H-3/ C-14 (δ C 174.9) and H-6/C-4 indicates the presence of a carboxyl group at C-4. Finally, the HMBC correlations of H-13/ C-7 (δ C 43.9), C-11 (δ C 154.7), and C-12 (δ C 65.0) demonstrate that C-7 is connected with acryl alcohol. Thus, the planar structure of 1 was assigned ( Figure 1). However, the ROSEY data cannot provide the correlation of H 3 -15/H 2 -6 ( Figure 3), which results in ambiguity in the relative configuration assignment of 1. Thus, NMR chemical shift calculations and ECD calculations were used to confirm the relative and absolute configuration of 1. More specifically, NMR calculations were carried out at the PCM/mPW1PW91/6-311+G(d,p) [16] level for (7R*,10S*)-1 (1a) and (7S*,10S*)-1 (1b), which are possible diastereomers of 1. The results reveal that 1a has the highest probability score. Next, ECD calculations on (7R,10S)-1 and (7S,10R)-1 were conducted according to the results obtained from NMR calculations. The CD spectrum matched well with the calculated ECD spectrum of 1a (Figure 4), revealing the absolute configuration of 1 to be 7R,10S, and it was named aquisinenoid F. Compound 2 was isolated as pale yellow gum, and was assigned the molecular formula C 15    of unsaturation. Compound 2 is similar in structure to 1 by analysis of their NMR data. There are two differences between 2 and 1. One is that at C-4 in 2 a methyl group is attached instead of a carboxyl group as in 1, which is supported by the HMBC correlations of H-3/C-4 (δ C 126.6), C-5 (δ C 134.4), and C-14 (δ C 19.5). The other difference is the existence of 8-OH in 2, which is confirmed by downfield chemical shifts at δ H 3.88 and δ C 71.0 and the key HMBC correlations of H-8/C-7 and C-9. Hence, the planer structure of 2 was obtained ( Figure 1). The Compound 3 was isolated as pale yellow gum, and its molecular formula was determined to be C 15   H 3 -15/Hb-6 allowed us to conclude that H 3 -15 and H-7 are in the opposite orientation. Thus, the relative configurations of chiral centers in 3 apart from C-11 were assigned. To determine the configuration of C-11 we performed NMR calculations. The results disclose that 3 has likely the configuration of (7R*,10R*,11S*)-3 based on the DP4+ probability analysis and the correlation coefficient. To clarify the absolute configuration of 3, ECD calculations were carried out. The spectrum of (7R,10R,11S)-3 ( Figure 4) agreed with the experimental spectrum, suggesting the 7R,10R,11S configuration of 3. As a result, the structure of 3 was determined as shown ( Figure 1) and named aquisinenoid H.  Figure 3) correlation of H 3 -16/Hb-6 indicated that its relative configuration was (5R*,7R*,10R*)-4. To elucidate its absolute configuration, the CD spectrum was determined and compared with the calculated spectrum. The results show that the calculated ECD spectrum of (5R*,7R*,10R*)-4 ( Figure 4) matches well with the experimental spectrum, suggesting that the absolute configuration of 4 is 5R,7R,10R. As a result, the absolute configuration of 4 was finally confirmed, and it was named aquisinenoid I. The 1D NMR spectra of 5 exhibit a pattern analogous to that of 3 ( Table 3). The difference is that the carboxylic acid at C-4 in 3 undergoes reduction to form a hydroxymethyl group at C-4 in
According to our previous studies, the components from A. sinensis possess various attractive bioactivities, such as antiinflammatory, anticancer, antirenal fibrosis, and acetylcholin-esterase inhibitory effects, which motivate us to assume that compounds with similar skeletons may have the same bioactivities. Therefore, the new compounds were evaluated for their anti-inflammatory and anticancer potential using the same method as described previously [16,22,23], and the cell viability was determined by CCK-8 assays (Figures A and B, Support-ing Information File 1). Unfortunately, we could not distinguish any one of these activities for all the new derivatives. Furthermore, we recall the skeletons in our current study and our previously reported ones [16,[22][23][24][25][26], which revealed the SAR. Moreover, eudesmane-type sesquiterpenes constructed with aldehyde groups are more active even in the form of dimers. In the present study, it was described that skeletons with 1°-alcohols and/or acid groups suppress activity, which was consistent with the A. sinensis literatures that 1°-alcohols and/or acid groups suppress activity [25,27], and concluded that eudesmane-type sesquiterpenes constructed with aldehyde groups are more active than alcohols or acids.

Conclusion
In summary, five new eudesmane-type sesquiterpenes compounds (1-5) and five known compounds (6-10) were isolated from agarwood of A. sinensis. The discovery of these new compounds enriches the structural diversity and complexity of sesquiterpenes derived from agarwood. Unfortunately, none of the new compounds exhibits biological activity against LPS-induced inflammation in Raw264.7 cells and human breast cancer cells. However, we have drawn good conclusions for SAR studies based on the current study and our previous study. These compounds will be isolated by other researchers in the future, who could consider our conclusions and choose other aspects of biological activity to study.

Plant material
The resinous wood of Aquilaria sinensis was purchased from Hainan Xiangshu Agarwood Industry Groud Co., Ltd., July 2018. The material was identified by the Gansu Institute for Drug Control, and a voucher specimen (CHYX0642) was deposited at School of Pharmaceutical Sciences, Shenzhen University, P.R. China.